1. Field of the Invention
The present invention relates to the field of tamoxifen derivatives and analogs, particularly halogenated tamoxifen derivatives and analogs. In that novel tamoxifen derivatives are described wherein the aliphatic chain of the molecule is substituted with a halogen group, the present invention also relates to methods of synthesizing tamoxifen analogs and derivatives.
In that the described tamoxifen derivatives have high affinity for binding estrogen receptors and may be labeled with detectable "tagging" molecules, rendering labeled estrogen receptors highly visible through positron emission topography (PET), single photon emission computed tomography (SPECT) and magnetic resonance imagining (MRI) the present invention also relates to reagents, radiopharmaceuticals and techniques in the field of molecular imaging.
The halogenated tamoxifen derivatives of the present invention are advantageously used in the imaging of estrogen receptors, for example, in breast, ovarian, uterine and brain tissue and may therefore be useful in the diagnosis of estrogen-receptor positive cancers, meningiomas and endometriosis.
The present invention also relates to the field of anti-cancer therapeutic agents, particularly to methods of breast tumor therapy, in that the described high affinity of these halogenated (i.e., iodo-, fluoro-, bromo- and chloro-) tamoxifen derivatives for estrogen receptors may be advantageously used to treat estrogen-receptor positive tumors.
2. Background of the Invention
Endocrine therapy provides an important nonsurgical method for treatment for breast carcinoma. This type of therapy is still considered standard for certain subsets of patients, typically postmenopausal women whose primary tumors have high estrogen levels..sup.1-3 The synthesis of F-18 fluoroestradiol for application in diagnosing breast tumors in humans has recently been described..sup.4 Observation of significant changes in the binding of estrogen receptors in breast tumors were reported using PET. However, technical difficulties associated with estrogen receptor saturation in patients receiving tamoxifen, or other estrogen receptor antagonist, has been observed to decrease the sensitivity and accuracy of using an estrogen-based receptor tag in diagnosing and monitoring the progress of tumors in patients receiving such treatments.
Tamoxifen (1), a potent non-steroidal antiestrogen, has been widely used in the treatment of human breast tumors. Tamoxifen has few side effects when compared with other hormonal treatments. Tamoxifen is cytostatic (i.e, it prevents/inhibits cell growth), and exerts competitive inhibitory activity at the receptor level with estrogen. More specifically, the cytostatic activity of tamoxifen results from its ability to bind to cytoplasmic estrogen receptors and be translocated to cell nuclei, where cell proliferation is prevented..sup.1-3 Thus, tamoxifen is often administered as an anticancer agent..sup.6 For example, Foster et al..sup.6 describes the effect of various tamoxifen hydroxy-derivatives on the growth of MCF-7 breast cancer cell line in its native form. However, highly active in vitro hydroxy tamoxifen derivatives were found to be less active than tamoxifen in vivo against a DMBA-induced ER-positive tumor in rats and only slightly more active against a hormone dependent mammary tumor in mice.
Tamoxifen has a relatively low binding affinity for the estrogen receptor (ER). Attempts have therefore been made to synthesize tamoxifen derivatives having improved ER binding affinity and specificity to enhance its action as an anti-cancer therapeutic agent. The structure of tamoxifen is demonstrated as: ##STR1##
A variety of modified tamoxifen derivatives have been described in the literature. Structural modifications have been made at virtually every site on the three aromatic rings of the tamoxifen molecule. For example, a 4-hydroxytamoxifen derivative in which X.dbd.--OH has been developed having the structure shown below .sup.33 : ##STR2##
However, while the 4-hydroxytamoxifen derivative was shown to be a potent anti-estrogen in vitro, it proved to be less effective than tamoxifen in vivo, owing to rapid glucuronidation of the hydroxyl group, followed by excretion. 4-Hydroxytamoxifen is the active intracellular form of the tamoxifen molecule in vivo, due to cytoplasmic hydroxylation after tamoxifen enters the cell. However, when 4-hydroxytamoxifen is administered in vivo, its polarity reduces its ability to cross the cell membrane, thereby reducing its access to estrogen receptors located in the cytoplasm. Therefore, in vivo tests indicate 4-hydroxytamoxifen to be less active than the native tamoxifen..sup.23
Other tamoxifen derivatives having a 4-position substitution of the phenyl ring, in which X is methoxy, methyl, fluoro or chloro, have also been proposed and evaluated..sup.15 K. E. Allen et al. (1980) conducted studies wherein the 4-methyl, 4-chloro and 4-fluoro derivatives were evaluated and found to have approximately equal activity for estrogen receptor binding affinity compared to tamoxifen in vitro. However, uterine weight tests indicated that these phenyl group derivatives had lower anti-estrogenic activity than tamoxifen, while other tests indicated that the activity of the 4-methoxy phenyl derivative was about the same as native tamoxifen.
A 4-iodo substitution of the phenyl ring as a tamoxifen derivative (formula 2: X=iodo) has recently been found to have greater potency than tamoxifen in relation to detecting estrogen receptor-positive breast cancer..sup.13 Other 3-iodo, 4-iodo, 3-bromo and 4-bromo phenyl ring-substituted tamoxifen derivatives have also been described..sup.13 For example, the McCaque et al. patent (U.S. Pat. No. 4,839,155) described the preparation of an iodo or bromo halogenated tamoxifen. However, the halogen, I or Br, was again substituted at one of the phenyl rings of the tamoxifen structure.
Derivatives of tamoxifen wherein other than the phenyl groups of the molecule are substituted have not been proposed in the art. Such a molecule would be desirable, as it would leave the major portion of the molecule unchanged and free to bind with the "target" molecule or tissue cells. Additionally, to further enhance tissue targeting specificity, a non-phenyl ring halogenated tamoxifen derivative would preferably be coupled with a "targeting" molecule, such as a microparticle.
Non-phenyl ring halogenated tamoxifen derivatives with enhanced binding affinity, greater specific radioactivity, and which can readily traverse the cell membrane have not as yet been developed in the art. The development of such derivatives would represent a tremendous improvement in the quality of imaging techniques currently available, as well as improve the accuracy of PET and SPECT scans.
Other alternative compounds proposed as possible radiopharmaceuticals useful in the imaging of tissue receptors include labeled progesterone and estrogen derivatives. For example, Pomper et al. described a ligand for the progesterone receptor..sup.16 The aliphatic fluorination of FENP (21-[.sup.18 F]fluoro-16-.alpha.-ethyl-19-norprogesterone) is described as demonstrating a high specific uterine target tissue uptake..sup.16 This ligand for the progesterone receptor was labeled with the positron-emitting radionucleotide fluorine-18 (t 1/2=110 min).
Estrogen-based imaging agents described in the literature include radionuclides of iodine.sup.20, fluorine.sup.19, and bromine.sup.21. By way of example, an estrogen-based imaging agent described in the literature is the 16-.alpha.-[.sup.18 F]fluoro-17-.beta.-estradiol ligand..sup.17
The preparation of 16-.alpha.-[.sup.18 F]fluoroestrogens and their selective uptake by estrogen target tissues in rats has been described by Kiesewetter et al..sup.19. Significant changes in the binding of estrogen receptors in breast tumor were reported with the use of [.sup.18 F]fluoroestradiol using PET..sup.4 However, the radioisotope .sup.18 F has a very short half life, and therefore techniques and molecules which employ this radioisotope must be rapid, and preferably more rapid than currently employed molecular labeling techniques allow.
Unfortunately, estrogen-based imaging agents are of limited utility in patients receiving estrogen based therapies due to the competition between imaging agents and therapeutic agents for estrogen receptors. Thus, a poor correlation is likely to exist between the actual physiological response within the tumor during hormonal therapy versus the response which is shown by an estrogen-based imaging agent. For these reasons, a progestin-based imaging agent for breast tumors might be preferred over an estrogen-based agent because tumor response to hormonal therapy appears to correlate better with progesterone receptor positivity than with estrogen receptor positivity..sup.17 It has further been reported that estrogen receptor positive tumors in patients on hormonal therapy (e.g. tamoxifen) could not be imaged with an estrogen, as the circulating levels of tamoxifen and its metabolites are sufficiently high to fully occupy the estrogen receptor.sup.18, making visualization quite difficult.
While the radiolabeled tamoxifen derivatives described in the literature have demonstrated some increase in estrogen receptor binding affinity, they do not demonstrate sufficient specific radioactivity due to the low tamoxifen phenolic ring incorporation of the radioactive halogen atoms. Thus, the derivatives' enhanced affinity for estrogen receptor is offset by a reduction in the radioactivity incorporated.
Moreover, the fluorine ion radioisotope, .sup.18 F, with its reportedly low effective dose equivalency and a short half-life (t 1/2=110 min) further exacerbates the problem of obtaining sufficiently labeled reagent, which is stable over an experimentally useful period of time.
For these reasons, any method which would utilize .sup.18 F in labeling the phenyl rings of tamoxifen molecule must be rapid (i.e. within a 2 hour reaction time) to avoid a loss in specific activity of the label.
Currently used tamoxifen derivatives, substituted at the various phenolic sites of the tamoxifen structure, can potentially block the formation of the active metabolite, 4-hydroxytamoxifen. Such a blockage may result in a decrease in receptor binding affinity of the particular tamoxifen analog since the 4-hydroxylated derivative is known to possess higher affinity. Alternatively, a competitive elimination reaction of 4-position substituted analogs may occur in the cytosol through the formation of the active metabolite, 4-hydroxytamoxifen. Such elimination processes are known to sometimes occur after drugs cross cell membranes.
Tamoxifen derivatives which could be more rapidly synthesized, with higher specific radioactivity and/or with improved receptor binding affinity or specificity, would offer a significant advance to the art, especially with regard to the in vivo diagnosis and therapy of estrogen positive tumors and the imaging of estrogen receptors in patients on a hormone-based regimen.
Numerous studies have shown that retinoic acid (RA) provides a promising new approach to the prevention and treatment of cancer. For instance, RA has been used as a clinically effective treatment for promyelocytic leukemia (PML) and juvenile myelogenous leukemia (JCML) in a majority of patients. RA is also active against papillomas, squamous cell carcinoma and other skin diseases (e.g., acne, psoriasis). It was hypothesized that these disorders may be due to the abnormal gene expression of RA receptors. Two subtypes of RA receptors, RARs and RXRs, are important in the biological actions. RA receptors may act to up-regulate gap junctional communication, stabilize normal cells by increasing the secretion of TGF-.beta. (transforming growth factor) against subsequent transformation and thus, decrease cell proliferation.
RA is capable of controlling gene expression, yet, the use of RA in therapy has been hampered by its high toxicity and teratogenicity, which may be associated with its lipophilicity. Therefore, a more hydrophilic RA analogue needs to be developed that may be used for chemoprevention and as chemotherapeutic agents.
Endocrine therapy, one of the oldest nonsurgical methods for the treatment of breast cancer, is still considered standard for certain subsets of patients, typically, postmenopausal women whose breast tumors have higher levels of estrogen receptors. The presence of sex hormone receptors in both primary and secondary breast tumors is an important indicator both for prognosis and choice of therapy for the disease. Tamoxifen, a potent antiestrogen that binds to cytoplasmic estrogen receptors and prevents cancer cell proliferation, has been widely used in the therapy of ER(+) breast tumors. Compared with other hormonal treatments, tamoxifen has few side effects. Tamoxifen therapy results are positive in 30% of unselected patients with breast cancer. In patients with ER(+) tumors, a response rate of 50% to 60% was obtained. Patients with metastatic cancer who do respond to the treatment have a response duration of 10 to 18 months and prolonged survival.
It has been shown that the concentrations of serum selenium and Vitamins A, C, and E were increased significantly in patients treated with tamoxifen for 3-6 months. The results suggest that tamoxifen therapy exerts significant positive effects on the rate of lipid peroxidation and protective systems in postmenopausal women with breast cancer. In the cancerous stress condition, the requirement for vitamins and antioxidants increases progressively, therefore, the level of vitamins decreased in women with untreated breast cancer as opposed to normal control subjects. Thus, it would be advantageous to provide an anti-cancer therapy which prevents depletion of vitamins.